The detection of bacteria and their accurate identification play a very important role in clinical diagnostics. Thus, a particular pathogen in bacterial infections should be identified as quickly as possible in order to be able to initiate an appropriate treatment.
In this context, Gram-positive bacteria, in particular mycobacteria, play an important part. Currently more than 100 mycobacterial species are known which have been detected and differentiated from clinical material. Pathogenic mycobacterial species such as the Mycobacterium tuberculosis complex, Mycobacterium intracellulare and Mycobacterium avium are of particular importance here.
In medical routine diagnostics and in food analysis, bacteria have traditionally been identified via selective media and subsequent investigation of the biochemical properties. Frequently, however, it is not possible here to determine the exact species. Moreover, these studies are very time-consuming, and a pure culture must be present for more extensive studies. Highly complex and diverse groups of bacteria with difficult growth conditions in particular are difficult to access by traditional culture differentiation and/or slow down diagnostics considerably due to their slow growth.
In recent years, nucleic acid-based methods have increasingly been introduced in order to detect bacteria. Said methods comprise, for example, carrying out nucleic acid amplification reactions using species-specific primers. Detection is usually via gel electrophoresis or via immobilized probes in microtiter plates. However, such techniques are not suitable for detecting one or more out of a multiplicity of possible pathogenic organisms. One approach to this problem is an amplification reaction mixture containing a mixture of different species-specific amplification primers and corresponding probes and/or a primer pair complementary to a base section of a group of organisms. The species specificity is ensured here via the structure of the probes.
The ribosomal RNA (rRNA) or ribosomal DNA (rDNA) including its spacer structures has already been used as target sequence for amplification of bacteria-specific nucleic acid. The target sequence most frequently used diagnostically is 16S rRNA. It is the best represented sequence in the appropriate databases. However, due to the relatively conserved character, species-characteristic sequence structures are not always found. In contrast, the 16S-23S rDNA spacer region is highly variable within many species and well suited to identifying bacteria. It is also superior to the 16S rRNA with respect to its structural information content. However, these target regions have also only limited suitability for distinguishing between a large number of mycobacterial species, since their sequence variability is too high and thus the sensitivity to probes against said target region decreases.
Liesack et al. (1991) FEBS Lett. 281, 114-118 disclose the complete nucleotide sequence of Mycobacterium leprae 23S and 5S rRNA and various target sequences for detection of bacteria. A probe of the Helix 54 region for detecting Mycobacterium leprae was disclosed, inter alia. This, however, does not allow the conclusion that it is possible to distinguish between a very large number of different species of Gram-positive bacteria with high GC content, for example mycobacteria, on the basis of the Helix 54 target region.